IgG-Complement Adsorption Behavior Activates Macrophage Mediated Early Immune Responses on Zirconia Implants

Zirconium implants have gained popularity among clinicians due to their superior mechanical properties. However, zirconium implants usually perform less well in early osseointegration than titanium implants. And the degree of severity of the acute inflammation resulting from macrophage activation after implantation determines the result of the implantation. The mechanism by which zirconia implants cause more acute inflammation compared to titanium implants is currently unknown. Here, the complement activation on zirconium oxide is demonstrated, which causes differences in inflammation compared to titanium oxide. More adsorption of immunoglobulin G (IgG) and complement protein C1q together with the more efficient triggering of the complement system is shown to occur on ZrO₂ surfaces. Molecular dynamics (MD) simulations further reveal that IgG exhibits more accessible binding sites on ZrO₂ surfaces due to its hydrophobicity, leading to more efficient complement activation. Reduced inflammation of hydrophilized ZrO₂ compared to non-treated ZrO₂ demonstrates the role of hydrophobicity in the higher inflammation of ZrO₂. The results reveal that complement activation due to conformational changes and greater adsorption of IgG and C1q on ZrO₂ triggers inflammation caused by macrophages, providing new insights for implant design and performance optimization.     

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine**

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay‘s potential for epigenetic evaluation of clinical samples, benefiting research and patient management.     

Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogonality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair Ava^N/Npu^C can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.